During endocytosis in mammalian cells, the orderly traffic of receptors and the nutrients, hormones, growth factors they interalize depends on the acidic pH found in organelles of the endocytic pathway. Low pH in endocytic vesicles also plays a critical role in mediating the penetration of many important pathogens including viruses, bacterial and protozoan parasites and bacterial toxins; acidification may also help to generate the immune response against these and other foreign antigens. As in the previous grant period, the long-term objective of the proposed research is to characterize the mechanism and functions of acidification during endocytosis. Emphasis will be placed on studying the properties of endosomes, the organelles in which the dissociation and sorting of receptors and ligands occurs. In addition, attention will be devoted to the relationship between acidification and such processes as infection by intracellular parasites and processing of antigen-presenting cells. Specific objectives include: (1) Characterization of the mechanisms regulating the electrogenic proton ATPase of endosomes using organelles purified by free flow electrophoresis--a technique developed during the previous grant period; of particular interest will be the acidification of coated vesicles and endosomes occurring "early" in the endocytic pathway; reconstitution of the proton pump into proteoliposomes is also planned, to test the suggested involvement of other ion transporters (e.g., Na+,K+- ATPase) in limiting the acidification of early endosomes; (2) Identification of the distribution, morphology, and internal pH of endosomes labeled at different stages of the endocytic pathway; this will be accomplished by monitoring the pH-induced conformation changes of viral spike glycoproteins both biochemically and by immunocytochemistry; (3) Investigation of the acidification of vesicles involved in transcytosis across epithelial cells; these experiments will use transport vesicles isolated from liver or MDCK cells (transfected with transcytotic receptors) after selective labeling with pH-sensitive endocytic probes; (4) Characterization of how protozoan parasites such as Toxoplasma gondii inhibit acidification and lysosomal fusion of parasite-containing vacuoles; vacuole function will be monitored in intact cells (by immunofluorescence) and in vitro; (5) Investigate the role of endosomes in the processing of exogenous antigens by antigen-presenting cells; these studies will involve the isolation of endosomes from cells expressing class II MHC antigens following the endocytosis of various antigens; these will include enveloped viruses whose uptake was characterized during the previous grant period; using a "cell-free" assay for antigen presentation, we will investigate the intracellular site at which immunogenic MHC-antigen complexes are formed; intracellular transport of MHC molecules, and the role of invariant chain in MHC traffic, will also be studied.